Hi, This sort of problem can occur if you are missing your lowest resolution data and/or your model for the bulk solvent is inappropriate. You might want to double check these issues.
With 80% solvent you have to be careful when choosing your contour level. If you are a fan of normalized maps and contouring based on "sigma" (and it's not a sigma by the way) you should be aware that the normalization factor is calculated over the protein and all that empty space and will be smaller than one calculated for equivalent protein density in a low solvent crystal. Plus/minus 3 contours will be lower and the significance of features will be inflated. One way to calibrate a contour level would be to leave out a known good bit of model and calculate your difference map. Then select a contour level that shows the understood omission well. Other peaks that show up at that level are errors as significant as the one you created. Dale Tronrud On 01/28/11 12:29, Todd Geders wrote: > Greetings CCP4bb, > > *Short version: > > Very noisy difference maps from a crystal with extremely high solvent > content, seeking advice on how best to handle such high solvent content > to eliminate noise in difference maps. > * > *http://strayxray.com/images/coot.jpg* > > Long version: > > I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent > content. The space group is P4132 and I'm quite confident the high > solvent content is real (there is a species-specific set of helices > extending into the solvent channels that appears to prevent tighter > packing). > > I was able to get a MR solution using a structurally related enzyme, but > the difference maps are terribly noisy (see link). There are lots of > negative density in empty spaces between well-defined 2Fo-Fc electron > density. > > http://strayxray.com/images/coot.jpg > > The 2Fo-Fc density actually looks fairly good. The initial MR maps had > clear density correlating to the sequence differences between the MR > model and the crystallized protein. After fixing the model as best I > could, the refinement statistics are R/Rfree of 27.5/30.3 with a > data/parameters ratio of 1.7. > > The mosaicity ranges from 0.15-0.3, data were collected with 0.5° > oscillations and 180° of data were collected. > > http://strayxray.com/images/diffraction.jpg > > Since the crystals appeared to suffer from radiation decay (based on > scaling statistics), I only use the first 40° of data (which still gives > around 8-fold redundancy). Using more minimal wedges of data or more > data does not noticeably make better or noisier maps. > > Any advice on improving the maps? Could the noisy maps be due to the > extraordinarily high solvent content? > > I'd appreciate any advice or comments. > > ~Todd Geders
